The synthesis of two versatile building blocks for supramolecular anion binding motifs, 5‐(N‐Boc‐guanidinocarbonyl)‐1H‐pyrrole‐2‐carboxylic acid (1) and 5‐(N‐Cbz‐guanidinocarbonyl)‐1H‐pyrrole‐2‐carboxylic acid (2) is reported. Using these building blocks, a guanidiniocarbonyl‐pyrrole anion binding site can easily be introduced into more complex molecules by using standard amide coupling conditions. Both syntheses can be performed on a multi‐gram scale. The products are obtained in pure form and can be stored as solids without decomposition. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

[reaction: see text] N'-Substituted guanidiniocarbonyl pyrroles 7 were synthesized for the first time by activation of a Boc-protected guanidiniocarbonyl pyrrole 3 with triflic anhydride and subsequent reaction with a primary amine. These guanidinium cations are efficient receptors for the complexation of amino acid carboxylates even in water (K(assoc) > 10(3) M(-1)) as could be shown by UV titration studies.

The syntheses of several bis-cations 1-5 with a simple primary ammonium cation attached via flexible linkers of varying length to a guanidiniocarbonyl pyrrole oxo anion binding site are reported. In UV-binding studies in aqueous buffer solution these bis-cations showed efficient binding of various N-acetyl amino acid carboxylates. However, complex affinity is significantly depending on both the anion and the length of the linker in the bis-cation. With increasing linker length, complex stability first increases until an optimum is reached for bis-cation 3 with a C4-linker. Then the complex stability decreases again. The best binding substrate in this series is N-acetyl phenyl alanine, most likely due to additional cation-pi-interactions between the aromatic ring and the guanidiniocarbonyl pyrrole cation. The formation of the complex between bis-cation 3 and N-acetyl phenyl alanine carboxylate was investigated further by fluorescence titrations and NOE studies, as well as molecular mechanics calculations.

In order to further clarify if and how much ozone is generated during high-voltage leak detection and to identify measures to reduce the impact of ozone generation on product quality, a highly sensitive analytical system was employed to investigate the generation of ozone at different operational conditions of high-voltage leak detection integrity testing. The analytical system is based on oxidation of Iodide ions in solution and identification of the Iodine formed by N, N-Diethyl-p-phenylendiamine (DPD) according to DIN 38403. Sensitivity of the system was found suitable to detect ozone levels as low as 0.025 ppm (mg/L). High-voltage leak detection process parameters-inspection speed, high voltage, filling level of the ampoule, and exposure time to the ampoule to high voltage-were varied between maximum and minimum values applicable in integrity testing of different ampoule sizes. For variation of exposure time, ampoules were repetitively tested by the leak testing machine to achieve a maximum exposure time of the ampoule up to 24 s (exposure time during production ≤2.4 s). No ozone was detected during the study under all inspection conditions. Even repeated exposure of the ampoules to high-voltage leak detection did not result in generation of measurable ozone levels. It has to be concluded that high voltage leak detection is not prone to causing oxidation of the drug products. For drug products in ampoules, 100% integrity testing is requested both by European and US regulations. Detection of integrity defects like small holes or cracks in the ampoule can be performed by numerous methods. Due to automation requirements, only three methods have been used during routine production-vacuum decay, head space infrared spectroscopy, and high-voltage leak detection. High-voltage leak detection is the most common method used in the pharmaceutical industry for 100% integrity testing of ampoules, but resent publications showed considerable oxidation of an air-sensitive active pharmaceutical ingredient during the integrity testing process. It was assumed that ozone generated in the inside of the vial due to exposure of the product to the high voltage during the testing process may be responsible for this result. As ozone is a very aggressive and non-selective oxidant, this result posed a serious thread not only to this product but also to the quality of many other products subjected to integrity testing by high-voltage leak detection.